The introduction of a foreign (non-self) substance, i.e. an antigen, to the immune system of a vertebrate usually results in the induction of an immune response by the host against that antigen. Typically this will involve the stimulation of B and/or T-lymphocytes, and the production of immunoglobulin molecules (antibodies) that recognise and bind to the antigen. There are a great many factors that influence the extent to which a substance will induce an immune response in a host. The degree of foreignness is important as the immune system has evolved and developed to be non-responsive to ‘self’. Size is also a significant factor, with larger molecules generally being more immunogenic than smaller ones. Molecules below ˜1000 Da molecular weight (classified as haptens) are too small to be seen by the immune system in isolation and are therefore non-immunogenic, though they may still be antigenic.
Larger molecules will be more complex and therefore more likely to contain multiple immunogenic epitopes, and are also more readily engulfed and processed by antigen presenting cells (APCs). The composition of the substance is also important, with proteins easily being the most immunogenic. Polysaccharides are much less immunogenic (in isolation) and nucleic acids and lipids are essentially non-immunogenic. Similarly, particulate or denatured antigens are more immunogenic than soluble and native molecules. The route of exposure and biological activities of foreign substances can also significantly affect the nature and extend of any immune response by the host. For example, parenteral injection of a substance that interacts with components or cells of the immune system will result in a much stronger response than mucosal exposure (ingestion/inhalation) of a relatively inert or inactive substance.
T-cells and B-cells recognise and respond to foreign antigens in different ways. Specialised antigen presenting cells or APC's (macrophages, dendritic cells and B-cells) continually interrogate their environment by taking up molecules from the extracellular space, including macro-molecules and whole micro-organisms, and processing the protein content of these. Exogenous proteins are digested by a panel of protease enzymes in endosomes, and the resulting peptides displayed on the surface of the cells in the groove of MHC II molecules. These in turn are recognised by specialist receptors on the surface of T-cells (TCRs). The process of T-cell development ensures that those T-cells displaying receptors that react to MHC II containing self-peptides are depleted, and only those that recognise foreign sequences mature successfully. The peptides recognised by T-cells (T-cell epitopes) are invariably linear, but are not always exposed or accessible on the native folded protein from which they were derived.
In contrast, the B-cell surface receptors or immunoglobulins (BCR) recognise and interact primarily with soluble proteins (both conformational and denatured epitopes), haptens, polysaccharides, and to a lesser extent some lipids and nucleic acids. The specificity of a BCR is identical to that of the antibody that the B-cell can secrete. Upon binding of its cognate antigen, the BCR is internalised and the bound antigen processed. Only when it is a protein, or is attached to a protein component, will it then be presented on the cell surface as part of an MHC II complex. Under these conditions, the B-cell is then available to be stimulated by a T-helper cell that has a TCR recognising the presented peptide. In the case of a large or complex protein, a B-cell can therefore be activated by a number of different T-cells, none of which need necessarily recognise the same antigenic epitope as the BCR, but all of which will recognise a peptide component of the same protein. It is this capacity of vertebrate immune systems that allows them to develop antibodies against antigenic determinants that are not in themselves immunogenic.
In order to develop effective vaccines, it is necessary to present antigenic epitopes to the host immune system in such a way as to stimulate a strong immune response, involving both T- and B-lymphocytes. Immune responses that do not involve activation of effector (helper) T-cells and subsequent stimulation of B-cells by these are usually short-lived and do not result in antigenic memory, i.e. do not lead to a more aggressive and more rapid antibody response when the host is exposed to the immunogen for a second time.
It is also often a requirement of vaccines to illicit antibodies that are able to inhibit, block or otherwise neutralize the functional activity of the target, and so afford protection to the host. This can present a major challenge for many reasons. Frequently, those epitopes that need to be targeted by an antibody response have not been identified due to a lack of structure-function data relating to the target. Even when detailed information relating to the target and its interactions, the identified epitope(s) may not be immuno-dominant and therefore might not generate the responses sought in the majority of patients. In other cases, the key protective antigenic determinants might not be protein, e.g. polysaccharides on pathogen glyco-proteins, and so not immunogenic (T-cell dependant) in isolation.
The vast majority of vaccines are delivered through parenteral routes; however there are many advantages to mucosal delivery such as patient compliance, self-administration, reduced risk of infection, and the possibility of inducing both mucosal and systemic immunity. There are also many obstacles to overcome, such as vaccine dilution, the presence of micro-flora, the need to withstand low pH when given orally, to cross membranes and the need for potent adjuvants (Vajdy et. al., 2004). Moreover, mucosal administration can lead to B-cell tolerance rather than an immune response. The dosage can also have a major effect on immune responses. If an immunogen is not effectively cleared by the immune system, or if the system is swamped by too high a dose, then tolerance can be induced. Conversely, too low a dose can also lead to tolerance, or simply fail to stimulate sufficient immune cells.
A number of approaches have been developed to help overcome these difficulties. In most cases, vaccines are administered along with some form of adjuvant. Adjuvants are essentially any formulation that, when administered together with an immunogen, causes one or more of persistence of the immunogen at the site of injection, enhancement of co-stimulatory signals, non-specific stimulation of lymphocyte proliferation or granuloma formation. They come in a variety of forms, for example Freund's complete adjuvant consists of inactivated Mycobacterium whilst others comprise an emulsion of oil (e.g. squalene) in water. These are most commonly used in animals as they can cause adverse reactions at the injection site. Some organic adjuvants are used in human vaccines such as Montanide© (mineral oil based with vegetable components) though more commonly they are inorganic such as aluminium salts.
Amongst the most popular and widely adopted methods to overcome low immunogenicity has been to couple an identified or desired antigen or antigenic determinant with a strongly immunogenic carrier. This is a protein or polypeptide derived from a different species, for example Bovine Serum Albumin (BSA) and Keyhole Limpet Hemocyanin (KLH) are often used as carriers of chemically conjugated haptens and small peptides to generate antibodies in animals (Berzofsky and Berzofsky 1993). The carrier presents the haptens on a molecule large enough to be seen and processed by the host immune system, and also stimulates the host immune response by being inherently immunogenic.
Generally speaking, carrier proteins derived from sources more phylogenically distant to the recipient are better. The carrier is then likely to be more different from host proteins and hence more foreign. A further important consideration when selecting a carrier protein is the possibility that if it is a homologue of a host protein, and so shares significant homology, then the elicited immune response might also react with host proteins and lead to adverse side effects. Non-protein antigens can only be coupled chemically, which may limit control over where on the carrier they are attached and how they are presented. Small peptides can be coupled chemically or genetically. In other areas of research, modern developments in bioinformatics have led to an increase in the rational design of immunogens, and in particular of peptides.
The concept of peptide vaccines is based on identification and chemical synthesis of B-cell and T-cell epitopes which are immunodominant and can induce specific immune responses, for example coupling a B-cell epitope of a target molecule to a widely recognised T-cell epitope to make it immunogenic (Naz R. K. and Dabir P. 2007). Peptides are seen as being relatively easy to produce when compared to larger and more complex protein antigens. They can also possess favourable chemical stability, and lack oncogenic or infectious potential making them attractive vaccine candidates. However, several obstacles limit the widespread usefulness of peptide vaccines including their often low inherent immunogenicity and the need for better adjuvants and carriers. Other research has suggested that recombinant chimeric proteins may be made more immunogenic if T helper epitopes are incorporated as tandem repeats (Kjerrulf M, et al. 1997).
Another popular class of carrier protein is bacterial toxoids. In the case of vaccines against bacterial infection where the symptoms of infection are caused by the action of toxins, then these can be used as the vaccine itself. It is of course necessary to render them inert, either chemically or by the use of a non-toxic component. Such attenuated toxins e.g. diphtheria and tetanus vaccines that were developed in the 20th century are called toxoids. Polysaccharide-protein conjugate vaccines in use or late stage development by companies such as Wyeth (Pfizer), Aventis Pasteur, GSK, Merck and others use tetanus, diphtheria or other toxoids.
The B sub unit of Cholera toxin or E. coli heat labile enterotoxin (LT) have been proposed by many as a useful carrier proteins for various vaccine applications (Nemchinov, L. G et al. 2000, George-Chandy, A. et al. 2001, U.S. Pat. No. 6,153,203). It is highly immunogenic, and in the absence of the CT-A sub unit is non-toxic. Forming the basis of a widely used Cholera vaccine it has a demonstrated safety profile when used systemically. Whilst relatively small (˜12 kDa), it can assemble into stable pentamers giving it a much higher molecular weight.
Of particular interest to many researchers is to exploit the affinity of CTB and enterotoxin pentamers to GM1 ganglioside, a branched penta-saccharide found on the surface of nucleated cells. During cholera infection, it is this binding that facilitates the translocation of the holotoxin across the intestinal epithelium. There have been numerous reports in the literature that vaccines based on CTB fusions, chemical or genetic, can be effective at stimulating mucosal immunity (George-Chandy, A. et al. 2001, Houhui Song et al. 2003, Shenghua Li et al. 2009, Harakuni, A. et al. 2005) when administered orally or intra-nasally. In order to retain the ability to react with GM1 ganglioside, which binds at the pocket formed between adjacent CTB subunits, it is essential that the target antigen does not block access to the GM1 binding site and does not prevent the assembly of CTB multimers.
It has been demonstrated that genetic fusions can be made to CTB that successfully retain GM1 binding, however there are also limitations. Liljeqvist, S. et al. (1997) showed that the serum albumin-binding domain of Strepococcal protein G could be fused genetically to either N- or C-terminus of CTB, or to both termini simultaneously, and retain GM1 binding. It was noted however that the N-terminal fusion and the dual fusion proteins were significantly less efficient at forming stable pentamers, and less effective in binding to GM1. Similarly, it has been demonstrated that large genetic fusions are unable to form pentamers unless a heterogenic mixture of both chimeric and wild type CTB are present (Harakuni, A. et al. 2005).